SEQUENCE-SPECIFIC ASSIGNMENTS OF THE BACKBONE H-1, C-13, AND N-15 RESONANCES OF THE MUTT ENZYME BY HETERONUCLEAR MULTIDIMENSIONAL NMR

被引:28
作者
ABEYGUNAWARDANA, C
WEBER, DJ
FRICK, DN
BESSMAN, MJ
MILDVAN, AS
机构
[1] JOHNS HOPKINS UNIV,SCH MED,DEPT BIOL CHEM,725 N WOLFE ST,BALTIMORE,MD 21205
[2] JOHNS HOPKINS UNIV,DEPT BIOL,BALTIMORE,MD 21218
关键词
D O I
10.1021/bi00211a017
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The MutT protein, a 129-residue enzyme from Escherichia coli which prevents A.T --> C.G mutations, catalyzes the hydrolysis of nucleoside triphosphates (NTP) to nucleoside monophosphates (NMP) and pyrophosphate [Bhatnagar, S. K., Bullions, L. C., & Bessman, M. J. (1991) J. Biol. Chem. 266, 9050-9054], by a mechanism involving nucleophilic substitution at the rarely attacked beta-phosphorus of NTP [Weber, D. J., Bhatnagar, S. K., Bullions, L. C., Bessman, M. J., & Mildvan, A. S. (1992a) J. Biol. Chem. 267, 16939-16942]. The bacterial MutT gene was inserted into the plasmid pET-11b under control of the T7 promoter and overexpressed in minimal media, permitting labeling of MutT with C-13 and/or N-15. The yield after purification of the soluble fraction was approximately 35 mg of homogeneous MutT/L with physical and enzymatic properties in distinguishable from those of the originally isolated enzyme. Essentially complete sequence-specific assignments of the backbone HN, N, Calpha, Halpha, and CO resonances of the free enzyme (1.5 mM) were made at pH 7.4 and 32-degrees-C, by heteronuclear double- and triple-resonance experiments using a modified Bruker AM 600 NMR spectrometer. Specifically, H-1[N-15]HSQC, H-1[N-15]TOCSY-HMQC, and H-1[N-15]NOESY-HMQC experiments were done with uniformly N-15-labeled enzyme. A H-1[N-15] HSQC experiment was done with selective [alpha-N-15]Lys-labeled enzyme. Also HNCA, HN(CO)CA, HNCO, constant time H-1[C-13]HSQC, HCACO, and HCA(CO)N experiments were done with uniformly C-13- and N-15-labeled enzyme. Sequence-specific assignments were initiated from HN and N-15 chemical shifts of Gly residues and of selectively labeled Lys residues in H-1[N-15]HSQC experiments. They were confirmed by Calpha chemical shifts of Ala residues uniquely identified by residual coupling to Cbeta resonances in constant time H-1[C-13]HSQC experiments. The sequence-specific assignments proceeded bidirectionally, terminating at Pro residues and at residues with undetectable NH signals, and the segments were linked to complete the backbone assignments. The backbone assignments reported here have permitted the interpretation of NOEs in the elucidation of the solution secondary structure of MutT, and the Calpha and Halpha chemical shifts have provided an independent approach to identifying secondary structural elements and to define their extent [Weber, D. J., Abeygunawardana, C., Bessman, M. J., & Mildvan, A. S. (1993) Biochemistry (following paper in this issue)].
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页码:13071 / 13080
页数:10
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